This project is designed to explore spectrin and associated cytoskeletal proteins in regard to the structure and function of the developing nervous system. During the first 2 years of this grant we have discovered two subtypes of brain spectrin (240/235) and (240/235E) in the mammalian brain, and localized these isoforms with immunocytochemistry and immunoelectron microscopy. Brain spectrin (240/235) is located primarily in the axons and presynaptic terminals of neurons. Brain spectrin (240/235E) is found in the cell bodies, dendrites, and postsynaptic terminals of neurons, as well as in certain glial cell types. The ontogeny of these spectrin subtypes was explored in immunoautoradiography experiments, and closely examined with immunocytochemical procedures. Each spectrin subtype had a distinct pattern of expression and distribution in the developing nervous system. In this application we describe experiments which are designed to give us a better understanding of brain spectrin subtypes and associated spectrin binding proteins in regard to the development of the mammalian nervous system. The aims of this proposal are: (1) Determine the location and distribution of brain spectrin subtypes in the developing mouse brain using immunoelectron microscopy. (2) Quantitate brain spectrin (240/235E) with quantitative immunodot assay. (3) Define whether there are structural changes in the isoforms of spectrin during development using immunoprecipitation and peptide mapping techniques. (4) Examine whether there are two ankyrin (syndein) subtypes in the mammalian brain utilizing immunoblots and immunohistochemistry. (5) Determine the ontogeny of ankyrin subtypes in the brain with immunoblots, immunodots, immunohistochemistry, and immunoelectron microscopy. (6) Examine the expression of amelin during mammalian brain development employing the techniques outlined in (5). The studies proposed constitute important and novel inquiries into the functional significance of spectrin-like proteins in the binding proteins in neural cells and tissues. This investigation is part of an ongoing program in cellular and molecular neurobiology which seeks to understand the process of normal and abnormal brain development.